DESCRIPTION: The goal of this proposal is to characterize the pathway of apoptosis. The approach of this proposal is to focus on the end result of apoptotic reactions, namely the activation of DNase-II and to define the potential effect of proteases and phosphatases, on the pathways of apoptosis. Apoptosis is a form of cell death induced as a consequence of many types of physiological, pathological, and cytotoxic insults. Results from this laboratory and others have shown that products of oncogenes and tumor suppressor genes can influence the apoptotic process and alter the outcome of therapeutic strategies. There is a major gap in the understanding of events occurring between the interaction with the primary cytotoxic targets and the eventual death of the cells. The approach of this laboratory is to identify events that occur in a dying cell and then to work back to the signals that regulate these events. The applicant focuses on DNA digestion as a frequent event occurring in a dying cell. Identification of the endonucleases involved will facilitate understanding of their activation by intracellular signals. Deoxyribonuclease-II (DNase II) has been identified as one of the endonucleases that potentially may be involved in apoptosis. DNase II cDNA has been cloned and antibody has been raised. The applicant proposes to perform studies in which DNase II will be either overexpressed or deleted in order to establish its role in apoptosis. Several isoforms of DNase II (30/31-, 26-, and 21-kDa isoforms) have been detected during apoptosis. The catalytic activity of these forms will be determined. The significance of endonuclease processing will be determined by genetic manipulation. The applicant proposes that intracellular acidification occurring during apoptosis may contribute to the apoptotic reaction. The acidification has been attributed to an alteration in the set-point of the Na+/H+ anti- port. The set-point is regulated by phosphorylation, suggesting protein kinases and phosphatases as possible regulators of apoptosis. Experiments have shown that inhibition of phosphorylation can protect cells from apoptosis. The applicant proposes to examine the regulation of protein phosphatase PP1, which has been implicated in the cascade of apoptosis. Proteases have also been implicated as regulators of apoptosis, consistent with the cleavage of DNase II. Markers of protease action (poly-ADP-ribose synthetase, terminin, and DNase II) and of phosphatase action (e.g. dephosphorylation of Rb) will be used to study this pathway. Inhibitors will be applied to each of the steps which will facilitate analysis of the temporal relationship between phosphatases, proteases, and endonucleases. Cell lines expressing bc1-2 will be used to locate steps that are modified by its action. Finally, the applicant will attempt to reconstitute apoptosis in vitro to enable the potential regulators of endonucleases and apoptosis to be determined.